This project is based on a unique decade-Iong University-Industry partnership in the development and production of stool diagnostic tests for enteric pathogens. Our goal is to produce rapid, sensitive, specific and cost-effective diagnostics appropriate for public health laboratories and point-of-care use for the highest priority Category B enteropathogens. Our hypothesis is that real-time multiplex PCR is the future of enteropathogen diagnostic tests for public health laboratories and that dipstick-formatted antigen detection tests are ideally suited for point-of-care use. Preliminary studies by the Program Investigators have resulted in FDA 510k-approved stool antigen detection tests for Category B agents Entamoeba, Giardia and Cryptosporidia. Project investigators have also pioneered methodology for DNA extraction from stool and therefore have the immediate capability to develop improved and cost-effective diagnostic tests for all 3 of the major biodefense Category B enteric protozoa threat agents. Project 1 will develop stool antigen detection dipstick tests for point-of-care diagnosis of Biodefense Category B Enteric Protozoa and Shiga toxin. Project 2 will develop real-time multiplex PCR using molecular beacons, FRET and/or Taqman probes for public health laboratory diagnosis of Biodefense Category B Enteropathogens. Project 3 will field validate the diagnostic tests produced by Projects 1 and 2 and will correlate protozoal genotype with virulence. The Laboratory Core will provide reagents, testing, and be the clearinghouse for obtaining, culturing and distributing Category B enteric parasites and bacteria for Projects 1-3. The Administrative Core will coordinate activities of the three projects, and facilitate scientific exchange and interactions of investigators. It will also provide administrative oversight and financial accountability. Successful completion of these studies will yield high throughput diagnostic tests that target multiple enteropathogens simultaneously in a single sample.